stress induction methods for establishing the chronic unpredictable mild stress rat model

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

Major depressive disorder (MDD) is a prevalent mental illness, ranking as the third leading cause of disability globally and affecting over 300 million people1,2,3. Notably, it is estimated that at least half of the affected individuals do not receive adequate treatment4. Given this gap, animal models serve as a crucial tool for investigating the etiology of depression. To date, more than 20 different animal models for depression exist5. Among these, the chronic unpredictable mild stress (CUMS) model, refined by Paul Winer in 1987, is the most frequently utilized6. The CUMS model operates on the premise that exposing rodents to a diverse range of socio-environmental stressors leads to symptoms akin to anxiety, tension, and depression. The methodology involves exposing animals to various mild stressors over several weeks, culminating in a range of behavioral alterations, including anhedonia and depressive-like behaviors7,8. These changes are accompanied by shifts in endocrine and neurotransmitter profiles, such as a reduction in 5-HT9,10. These outcomes closely mirror those observed in humans diagnosed with MDD, thereby validating the model's utility. The CUMS model is particularly valued for its effectiveness in evaluating antidepressants, manifesting high levels of surface, structural, and predictive validity11,12. Unlike other models, CUMS is sensitive to the effects of chronic administration of monoaminergic antidepressants. For instance, selective serotonin reuptake inhibitors (SSRIs) such as citalopram, paroxetine, and fluoxetine have been shown to prevent and reverse anhedonia under conditions of chronic stress12,13. Additionally, new rapid-acting antidepressants, such as ketamine, have also demonstrated efficacy in this model14,15. In contrast, other tests like the forced swimming test (FST) and tail suspension test (TST) are less reliable for modeling long-term behavioral changes, often reflecting adaptations to acute stress rather than enduring symptoms of depression16. These characteristics underscore the robust validity of the CUMS model in depression research. One of the most salient features of the CUMS model, recognized for its high reliability in classical studies, is anhedonia-the incapacity to experience pleasure or interest in everyday activities17,18. This phenomenon is commonly evaluated using sucrose preference tests, and many antidepressants have been shown to reverse reduced sucrose consumption. Several other metrics are also commonly employed in the CUMS literature, including the open field test (OFT), which assesses voluntary motor behavior, exploratory tendencies, and tension, thereby gauging the severity of depression19. Other tests such as the elevated plus maze (EPM) evaluate anxiety-like behaviors, the Morris water maze test (MWM) examines cognitive functioning20, and the FST assesses susceptibility to negative emotions and behavioral despair20. Furthermore, the majority of stressors that impact humans are social in nature. Individuals with suboptimal social relationships, characterized by limited social activities, networks, and support, are at a heightened risk for various diseases21,22. This is also relevant in the case of rodents, who are social animals living in groups. For example, rats that are housed in isolation exhibit characteristics of what is termed isolation syndrome, which induces social stress and expedites the onset of depression23.
Mongolian medicine, a significant branch of Chinese medicine, posits that the onset of disease is a complex interplay between intrinsic and external factors. These external factors, referred to as the four auxiliary conditions, encompass climate change, diet, lifestyle, and sudden events such as infections, startling incidents, and psychological disorders. The disease process is conceptualized as an ongoing interaction between three elements-referred to as the three types of homors-and the Seven Bodily Constituents in concert with the four auxiliary conditions24. Mongolian medicine holds that the human body functions as an integrated entity, maintained by a relative balance among the three homors. A disruption in this balance is considered to be a precursor to illness24. Given the pivotal role of animal experimentation in bridging traditional and modern medicine, it is crucial to develop animal models that are relevant for research in the field of Mongolian medicine. Accordingly, we employed a 28-day isolation methodology coupled with CUMS to simulate these physiological and psychological stressors. We selected nine specific unpredictable stressors and sought to underpin this modeling method through the Three homors theory of Mongolian medicine. Establishing a robust animal model is fundamental for advancing basic research in Mongolian medicine and will contribute significantly to its foundational studies.

Author Spotlight: Establishing a Rodent Model for Investigating Depression Factors in Traditional Mongolian Medicine

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

The scope of our research is Mongolian medical research. In this study, we are investigating the factors contributing to depression in Mongolian medicine, with a particular emphasis on the interaction between internal factors, such as wind, biofilm, and external factors. The problem of conforming to the lack of traditional Mongolian medical models was solved.This protocol addresses the establishment of an animal model based on Mongolian medical theory and then provides some suggestions for future research in Mongolian medicine. In a depressive disorder study, establishing an animal model is crucial for understanding new drugs'pathological mechanisms and effects. Compared with other techniques, this protocol is more in line with the pathogenesis and treatment principles of depression in traditional Mongolian medicine, which will help future research on the treatment of depression in Mongolian medicine.The protocol can demonstrate the relationship between external and internal factors interacting to produce disease and is in accordance with the theoretical system of Mongolian medicine. It will advance the basic research of traditional medicine on depression and provide some recommendations that will help subsequent researchers to research, develop, and utilize traditional medicines.

Stress Induction Methods for Establishing the Chronic Unpredictable Mild Stress Rat Model

After dividing the rats into a control, or con, and model, or mod, group, house them in individual cages. For the tail-clamp method, secure the tail of a rat and measure the clamping time for one minute. In the water deprivation method, remove the feeding bottle from the cage and record the rat for 24 hours.For the food deprivation method, remove the food from the cage and record the rat for 24 hours. For cold stimulation at four degrees Celsius, place a rat in a bucket of cold water and monitor its activity for five minutes. Then dry the rat with a blower and return it to its original cage.To administer 45 degrees Celsius heat stress, place the rat in an oven and monitor for five minutes. For the inversion of the light-dark cycle, wrap the cage in a black cloth for one hour to stimulate daytime darkness. Subsequently illuminate the cage for 12 hours at night to mimic daylight and record the data for 24 hours.For the wet padding experiment, pour 200 milliliters of water into a cage containing 100 grams of padding. Place the rat in the wet cage and monitor for 24 hours. After drying the rat as demonstrated earlier, return it to a cage containing fresh wood shavings.In the cage incline method, place the rat in a cage angled at 45 degrees against a wall and monitor it for 24 hours. For high speed shaking, place the rat in the mechanical shakers set at 160 RPM, and record data for 15 minutes. After applying stressors, transfer the mod group cage from the CUMS room to the housing room.

stress-induction-methods-for-establishing-chronic-unpredictable-mild

Research

JoVE Journal

Medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological ...

Behavioral Tests for Assessing Depression-Related Behaviors in Rats

After dividing the rats into a control, or a CON, and model, or MOD group, house them in individual cages. For the open field test, partition a black box into 25 square sections of equal area. Install a video tracking analysis system in the box.Place the rat in the central square and monitor its horizontal and vertical activities for five minutes. After the test, sanitize the box with 75%alcohol to remove any remaining rat odors. To assess anhedonia through the sucrose preference test, weigh bottle A containing water and bottle B containing 1%sucrose.Place both bottles on the cage lid and allow ad libitum access to both solutions for the rat. After consumption, weigh the bottles, and, using the given formula, calculate the 60 minute sucrose preference rates on given days. To perform the Morris water maze test, divide the pool into four quadrants, and number them from one to four.In the third quadrant, place a resting platform one centimeter below the water surface. Add milk to the pool to increase water opacity and maintain a water temperature of approximately 23 degrees Celsius throughout the experiment. Place the rat in one quadrant of the maze, and using the Morris Water Maze video tracking system, record the latency time to identify the platform for 120 seconds.Finally, remove the hidden platform from the pool. Then, place the rat into the water and record the number of zone crossings for 120 seconds. In the open field test, the MOD group exhibited lower vertical and horizontal scores compared to the CON group, indicating successful induction of anxiety.After 28 days, the MOD group showed a lower sucrose preference percentage than the CON group. The Morris water maze test revealed that the MOD group had a longer latency to locate the platform and crossed fewer times compared to the CON group. Further, the MOD group exhibited reduced hippocampal 5-HT levels compared to the CON group.